【作者】 阮妙鸿；

【导师】 张木清；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2007， 博士

【摘要】 甘蔗花叶病严重影响甘蔗生长，是甘蔗生产中主要病害之一。通过基因工程培育抗花叶病品种是甘蔗高效育种的辅助手段，2003年农业部甘蔗生理生态与遗传改良重点实验室通过基因枪介导将ScMV-CP基因转入高感花叶病的甘蔗品种拔地拉（Badila），获得了13个转ScMV-CP基因甘蔗无性系。本研究以转CP基因甘蔗作为材料，研究转基因甘蔗的抗病稳定性和工农艺性状表现；通过转基因甘蔗的光合特性研究其增产的光合基础；通过反复抗性鉴定，从中选择抗病强转基因无性系进行人工接种研究其抗病的生理生化基础和分子基础；并对转基因甘蔗环境安全性进行了初步评价；研究结果表明：转基因甘蔗明显提高了抗甘蔗花叶病毒的能力，人工接种和自然诱发鉴定，花叶病发病率在0-34％之间，而且在不同世代保持稳定的水平；而非转基因甘蔗拔地拉（Badila）发病率达92-100％；通过工农艺性状和抗病性综合考察，选出了5个转基因甘蔗进入环境释放，结果表明：转基因甘蔗发病率在0-20％之间，株高比非转基因甘蔗增加23-32％，有效茎数增加45-55％，每公顷蔗茎产量增加62-80.6％，甘蔗糖分绝对值提高1.15-2.15％。在13个转CP基因甘蔗中以转基因甘蔗B48表现最为优异，在多年的人工抗性鉴定和自然诱发鉴定均未发病，综合农艺性状表现优异。通过转基因甘蔗B48与非转基因甘蔗叶片细胞超微结构、叶绿体蛋白质组及叶片光合特性的比较，结果表明：非转基因甘蔗的叶片细胞超微结构呈典型的甘蔗花叶病毒侵染后的病理特征；叶片中细胞叶绿体被破坏，基粒片层消失；通过叶绿体蛋白的双向电泳进一步发现，非基因甘蔗叶绿体光系统Ⅰ、光系统Ⅱ中光系统Ⅰ蛋白、放氧复合体（OEC）蛋白下调，说明病毒侵染对非转基因甘蔗叶绿体的光系统Ⅰ、光系统Ⅱ造成损伤，从而使非转基因甘蔗叶片的CO₂同化能力下降；而转基因甘蔗的叶片细胞保持完整，细胞内未发现病毒粒子，叶绿体结构清晰，光系统保持正常，叶绿素含量高，PEPC酶活性强，从而使转基因甘蔗保持较高光合作用能力。接种甘蔗花叶病毒后，转基因甘蔗接种后游离SA增加的量比原种大，时间提前，同时SOD、POD活性提高，CAT活性显著降低，这有利于增加体内H₂O₂的积累，加强了细胞壁的氧化交联，限制了病毒进一步侵染；而非转基因甘蔗SOD、POD活性提高缓慢，下降快，CAT活性下降慢，影响了H₂O₂的积累，无法及时有效地建立抗病信号的传导途径来诱导抗性基因的表达。苯丙烷代谢研究结果表明，转基因甘蔗在接种后叶片PAL活性迅速上升，并保持相对较高的水平；而转基因甘蔗PAL活性上升要滞后于转基因甘蔗，而且下降迅速。非转基因甘蔗PPO活性上升比转基因甘蔗慢而且增加的幅度也小；转基因甘蔗接种后积累类黄酮速度快，持续时间长，含量增幅较大，在整个过程中保持一个相对较高的浓度，非转基因接种后类黄酮含量下降而且变动幅度大。利用本实验室开发的斑茅（甘蔗的近缘属植物）干旱胁迫下的cDNA微阵列，研究了转基因甘蔗B48和非转基因甘蔗接种后基因表达的差异情况，结果表明：转因甘蔗接种后上调表达的克隆有43个，主要参与细胞的结构保护、信号传导、蛋白质合成、能量代谢、呼吸作用等有关过程；这说明接种病毒激活了转基因甘蔗体内众多抗病基因。利用芯片杂交的结果，克隆了候选MYB基因的片段，生物信息学分析该MYB基因属于典型R2R3 MYB转录因子，实时定量PCR分析表明所克隆的甘蔗MYB基因在不同组织表达特异性不明显，但是受外源SA和H₂O₂强烈诱导。接种后的叶片蛋白质组研究表明：转基因甘蔗与非转基因甘蔗的叶片蛋白的2-DE图谱有明显差异，在转基因甘蔗叶片中有10个蛋白表达上调，1个蛋白特异诱导表达；而非转基因甘蔗中3个表达上调，1个特异诱导。质谱鉴定表明，转基因甘蔗中核因子激酶、细胞质APX和Mn-SOD被诱导上调表达；而在非转基因甘蔗中铁氧还蛋白和甘蔗旱诱导蛋白表达上调。转基因甘蔗环境安全性评价结果表明：田间转基因甘蔗与非转基因甘蔗害虫的危害没有差异，在土壤总DNA未检测到外源ScMV-CP基因；转基因甘蔗根际土壤的细菌和真菌数量明显提高，但放线菌数量差异不明显；转基因甘蔗中的NPTII标记基因并没有增加对卡那霉素抗性的土壤微生物数量；转基因甘蔗和非转基因甘蔗根际细菌多样性研究表明，Simpson指数（D’）、Shannon-Wiener指数（H’）、Mclntosh指数（DMc）差异不明显，说明转基因甘蔗对土壤细菌的多样性影响很小；转基因甘蔗显著提高了根际土壤脲酶活性，但对土壤的蔗糖酶和磷酸单脂酶没有影响。初步研究表明，转基因甘蔗对土壤细菌和真菌有一定的影响，但对放线菌影响很小；就目前来看，短期内对土壤肥力没有不利影响。更多还原

【Abstract】 Sugarcane mosaic disease is one of major disease to sugarcane and influences the growth of sugarcane seriously. Breeding resistant cultivars by genetic engineering is an assistant method in high-effective breeding of sugarcane. ScMV-CP gene was transformed to high-susceptible sugarcane cv. Badila by particle bombardment in the Key Lab of Sugarcane Eco-Physiology& Genetic Improvement, Ministry of Agriculture in 2003, and 13 ScMV-CP gene transgenic sugarcane were gained. Using these transgenic sugarcanes as material, the resistant stability and agronomic performance of transgenic sugarcane were studied after environmental release; and photosynthetic basic of yield increase was studied through the difference of photosynthetic characteristics between transgenic and non-transgenic sugarcane; After resistance identifying repeatedly, the high-resistant transgenic line was chosen to study the physiological and biochemical basis and molecular basic of resistance; and transgenic sugarcane environmental risk was assessed as well. The results showed as followed:Resistance to ScMV was improved significantly in CP transgenic sugarcanes, the incidence of ScMD（sugarcane mosaic disease） ranged from 0 to 34% after inoculation and natural infection, and kept stable in different generation of progenies. But the incidence of the non-transgenic sugarcane amounted to 92-100%. 5 transgenic lines were chosen for environmental release according to the agronomic performance and resistance of transgenic sugarcane, the results indicated that the incidence of ScMD ranged from 0 to 20%, stalk length increased by23-32%, effective stalk increased by 45-55%, cane yields of per hectare increased by 62-80.6%, and the sugar content（absolute value） increased by 1.15-2.15% in transgenic sugarcane compared with the counterpart in non-transgenic sugarcane. The transgenic line B48 was the best line in 13 transgenic sugarcane lines, and had nice agronomic traits and no disease development after inoculation or natural infection in four years.The cell ultrastructure, chloroplast proteome and photosynthetic traits in leaves of transgenic line B48 and the counterpart of non-transgenic line were studied. The results showed that the cell ultrastructure of non-transgenic sugarcane leaves displayed the typical cytopathological character infected by ScMV, and the cytoplasm was filled with ScMV particles, but none of these profiles were found in cell ultrstructure of transgenic sugarcane. These confirmed that the sugarcane mosaic disease was caused by ScMV and showed the disease resistance of the transgenic line on cellular level in addition. The chloroplasts were destroyed and the grana lamella disappeared in non-transgenic sugarcane leaves after infected by ScMV. The chloroplast proteome was studied by 2-DE. it was found that the chloroplast photosystem I protein and Oxygen evolution complex（OEC） of photosystem II（PS II） in leaves of non-transgenic sugarcane were down-regulated. These suggested that the PSI and PS II of chloroplasts in leaves of non-transgenic were damaged after virus infection, and resulted in the decrease of chloroplasts content and net photosynthetic ratc（Pn）. Moreover, the infection lowered the activity of phosphoenolpyruvate carboxylase （PEPC）. All of these changes in leaves of non-transgenic sugarcane decreased the CO₂ assimilation capacity and inhibited the plant growth, and finally caused the yield loss. While transgenic sugarcane had integrated cell structure and chloroplasts structure and accompanied with the high content of chloroplasts and activity of PEPC in leaves. So the transgenic line kept better capacity of photosynthesis. It can be regarded as the photosynthetic basis of the yield increase in transgenic sugarcanes.The content of endogenous free SA and conjugated SA increased both in transgenic and non-transgenic sugarcane after inoculation. And SA had a significantly negative correlation with CAT activity, increasing of SA inhibited the CAT activity and caused accumulation of H₂O₂. These were the common defense response to ScMV in transgenic and non-transgenic sugarcane. But the increase of free SA in transgenic sugarcane was higher than non-transgenic sugarcane, and ahead of the latter. At the same time SOD, POD activity increased and CAT activity lower remarkably in transgenic line. Changes of these enzyme activities leaded to accumulation of H₂O₂ and enhanced the cell wall cross—linking to limited the virus to infect further. But in non-transgenic sugarcane, the SOD,POD activity increased slowly, and decreased rapidly, and CAT decreased slowly. These changes of protective enzyme activities influenced the accumulation of H₂O₂ and cause the plant could not establish signal transduction pathways of resistance effectively.The studies of metabolism of phenylalanine showed the PAL, PPO activities enhanced both in transgenic sugarcane and non-transgenic sugarcane after inoculation with ScMV. PAL activity in transgenic line increased rapidly and kept stable after inoculation. But PAL activity in non-transgenic increased later than transgenic line and decreased promptly. And PPO activity in non-transgenic sugarcane was later and lower increase than transgenic sugarcane. The content of flavonoids in leaves accumulated fast, last a long time and kept a higher concentration in transgenic sugarcane after inoculation; But the content of flavonoids decrease and can not keep stable level in non-transgenic line. Metabolism of phenylalanine in transgenic sugarcane differed from non-transgenic sugarcane are cause by the different resistance to the ScMV.Different genes expression in transgenic and non-transgenic sugarcane was analyzed after inoculation used E. arundinaceum（sugarcane related plant） cDNA microarray under drought stress developed by our lab. The result showed there were 43 up-regulated clones in transgenic sugarcane. These clones took part in cell structure protection, signal transduction, protein synthesis, energy metabolism, respiration and so on. For example, cell wall protein GP2（HRGPGP2） and protein binding / structural constituent of cell wall played a role in cell structure protection, and zinc finger protein, MYB protein transcription factor took part in resistant signal transduction, and acetyl CoA carboxylase participated in secondary metabolism to produce flavonoids and other secondary metabolites. These results suggested that the resistance to ScMV in transgenic sugarcane due to resistant genes were activated rather than transferred CP gene into sugarcane. Candidate MYB gene fragment was cloned base on result of cDNA mircroarray hybridization. Bioimformatics analysis of the fragment suggested the sugarcane MYB gene belonged to the typical R2R3 MYB family genes. And result of real-time quantitative PCR analysis indicated the sugarcane MYB gene did not show any organ-specific, but induced by exogenous SA and H₂O₂ strongly. These suggested this MYB transcription factor of sugarcane may play a role in the resistance.Study on proteome in leaves of transgenic line B48 and non-transgenic line showed that images of 2-DE were very different from each other after inoculation. 10 proteins were up-regulated and 1 was induced specifically in leaves of transgenic sugarcane, but 3 were up-regulated and 1 was induced specifically in leaves of non-transgenic sugarcane. 7 different proteins were identified by MS/MS using 4700 Proteomics Analyzer （Applied Biosystems, USA）. The up-regulated proteins in leaves of transgenic sugarcane were IkappaB kinase, cytosolic ascorbate peroxidase （APX） and Mn-superoxide dismutase （Mn-SOD）, and unknown protein was specifically induced. The up-regulated proteins in leaves of non-transgenic sugarcane were unknown protein and rieske Fe-S precursor protein, and drought-inducible protein was specifically induced. These suggested that the different protein expression due to the different resistance of transgenic sugarcane and non-transgenic sugarcane.Environmental risk of ScMV-CP transgenic sugarcane was primarily assessed thought non-target organism investigation in the field, CP gene horizontal transfer to the soil microbe and effects on micro-ecology of rhizosphere soi in different growth stage of sugarcane and under different cultural patterns. Effects on micro-ecology of rhizosphere soil were studied using BIOLOG Ecoplate and plate cultivation of microorganism associated with related soil enzyme activities analyses. The results showed that there was no difference in pest harming between transgenic sugarcane and non-transgenic sugarcane, and no ScMV-CP gene was detected in soil total DNA; The total number of colony forming units（CFUs） of culturable bacteria and fungi were increased in transgenic sugarcane rhizosphere soil, but the total number of CFUs of culturable actinomycetes was not effected. Changes of CFUs of microbe resistant to kanamycin（Km） in medium indicated that number of soil microbe resistant to Km in soil wasn’t increased because of NPT II in transgenic sugarcane; BIOLOG experiment showed the metabolism activity of soil bacteria was increased lightly, and the soil bacteria diversity indexes such as Simpson index（D’）,Shannon-Wiener index（H’）, evenness, Mclntosh index（DMc） were not different between transgenic sugarcane and non-transgenic sugarcane after inoculating 72h in BIOLOG Ecoplate. Urease activities in the rhizosphere soil of transgenic sugarcane were significantly higher than that of non-transgenic plant, but sucrase activity and phosphatase activities were not influenced. These primary results indicated that transgenic sugarcane had some effects on soil bacteria and fungi, but on effect on actinomycetes; transgenic sugarcane had no disadvantage influence on soil fertility at the present time. These suggested changes of micro-ecology of rhizosphere soil were caused unlikely by exogenous gene expression in transgenic sugarcane, maybe was the non-prediction effect after environmental release of transgenic sugarcane.更多还原